title: Selective monitoring of ubiquitin signals with genetically encoded ubiquitin chain-specific sensors.
abstract: Despite intensive research, there is a distinct lack of methodology for visualizing endogenous ubiquitination in living cells. In this protocol, we describe how unique properties of ubiquitin (Ub)-binding domains (UBDs) can be used to selectively detect, visualize and inhibit Ub-dependent processes in mammalian cells. The procedure deals with designing and validating the binding selectivity of GFP-tagged K63- and linear-linked sensors (TAB2 NZF and NEMO UBAN, respectively) in vitro. We describe how these moieties can be used to inhibit tumor necrosis factor (TNF)-mediated NF-κB signaling and to detect ubiquitinated cytosolic Salmonella in living cells, emphasizing a more flexible use compared with chain-specific antibodies. These chain-specific sensors can be used to detect Ub-like or autophagy-related modifiers and, in combination with mass spectrometry, to identify new Ub targets. These Ub (-like) sensors can be designed, constructed and tested in ~2-3 weeks.
authors.affiliation: Institute of Biochemistry II, School of Medicine, Goethe University, Frankfurt am Main, Germany.
authors.forename: Sjoerd J L
authors.initials: SJ
authors.lastname: van Wijk
authors.forename: Evgenij
authors.initials: E
authors.lastname: Fiškin
authors.forename: Ivan
authors.initials: I
authors.lastname: Dikic
mesh_terms: D000818:Animals; D001665:Binding Sites; D015374:Biosensing Techniques / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016328:NF-kappa B / Q000378:metabolism; D017434:Protein Structure, Tertiary; D015203:Reproducibility of Results; D015398:Signal Transduction; D025801:Ubiquitin / Q000032:analysis* / Q000378:metabolism*
chemical_list: D016328:NF-kappa B; D025801:Ubiquitin; D049452:Green Fluorescent Proteins